Spinal cord injury (SCI) inflicts trauma to the cells and tissues of the central nervous system (CNS) and causes a severe and debilitating condition in the individual. Following SCI, limited regeneration of injured neurons results in permanent disability characterized by some loss of sensation, paralysis and autonomic dysfunction. One reason that neurons fail to regenerate is their inability to traverse the glial scar that develops following SCI. This glial scar contains extracellular matrix molecules including chondroitin sulfate proteoglycans (CSPGs). In vitro studies show that neurons fail to extend processes over CSPG coated surfaces, while in vivo data correlate failure of regeneration with areas of CSPG expression. Within the adult central nervous system (CNS) myelin there are also several identified axon growth inhibitor compounds like myelin associated glycoprotein (MAG), OMgp, and Reticulon 4 or Nogo that have been shown to be inhibitory for the growth of neurons.
The proteglycan degrading enzyme chondroitinase ABC type I has been used to enhance neuronal growth in a dorsal column lesion model of spinal cord injury. It has also been reported that treating a spinal cord injury with NOGO receptor antagonist promotes a certain limited degree of neuronal regeneration. It has further been reported that creating a NOGO knock out mouse resulted in certain limited and inconsistent degrees of neuronal regeneration following dorsal hemisection of the spinal cord.
Experimental treatments for injury to the CNS have utilized the application of chondroitinase to the extracellular space, however the enzyme digests CSPG in the extracellular matrix and not intracellular stores. Furthermore, diffusion of chondroitinase within the parenchyma the essential and distinctive tissue of an organ or an abnormal growth as distinguished from its supportive framework) or between anatomical compartments is limited. The limited access of drugs, imaging agents, and anesthetics, etc. to target cells and/or tissues of the Central Nervous System (CNS) may reduce the usefulness or effectiveness of any of these substances.
The delivery of therapeutic and diagnostic molecules into cells and tissues is, in part, dependent upon extracellular matrices as well as carbohydrates and proteins linked to cell membranes. The extracellular matrix is composed in part of proteoglycans, among them are the chondroitin sulfated proteoglycans (CSPGs). CSPGs are a family of proteoglycans composed of a core protein and covalently linked sulfated glycosaminoglycans. Each proteoglycan is determined by the glycosaminoglycan side chains. For CSPGs these side chains are made up of approximately 40 to 100 sulfated disaccharides composed of chondroitin 4, 6 and dermatan sulfates. The protein component of the CSPG is ribosomally synthesized and the glycosylation occurs in the endoplasmic reticulum and Golgi apparatus. The sugar chains are then sulfated at the 4 or 6 positions by several glycosaminoglycan sulfotransferases.
Transduction proteins may be used to transport polypeptides and polynucleotides cargo across anatomical barriers and into cells. For example, the TAT protein (SEQ ID NO: 2) from the human immunodeficiency virus (HIV) contains a protein transduction domain (PTD) that is involved in the transduction of HIV into cells. The PTD contains an 11 amino acid domain (TAT Peptide) (SEQ ID NO: 3) that is responsible for the activity of the PTD. The TAT Peptide (SEQ ID NO: 3) can be linked to proteins and facilitate the transduction of the proteins into cells. The mechanism of transduction is independent of the molecular weight or chemical properties of the proteins that are linked to the TAT Peptide (SEQ ID NO: 3). In vivo studies show that if a fusion protein consisting of the TAT Peptide (SEQ ID NO: 3) linked to the 120 kd enzyme, beta-galactosidase (β-Gal), is injected into mice, then a robust delivery of β-Gal into a wide variety of cells is observed. When linked to the TAT transduction peptide (SEQ ID NO: 3), β-Gal activity was observed in the brain; without the TAT Peptide (SEQ ID NO: 3), β-Gal was not observed in the brain. Transport across the blood brain barrier is normally restricted to certain hydrophobic small molecules and particular low molecular weight lipophilic peptides. Transport of proteins as large as β-Gal into the brain is usually not possible without substantial disruption of the blood brain barrier, but the TAT Peptide (SEQ ID NO: 3) facilitates transport while leaving the blood brain barrier intact.
Chimeric proteins, also called fusion proteins, are hybrid proteins which combine at least parts of two or more precursor proteins or polypeptides. Chimeric proteins may be produced by recombinant technology, i.e. by fusing at least a part of the coding sequence of one gene to at least a part of the coding sequence of another gene. The fused gene may then be used to transform a suitable organism which then expresses the fusion protein.
Tat Peptide Complexes Frankel et al. (U.S. Pat. Nos. 5,804,604; 5,747,641; 5,674,980; 5,670,617; 5,652,122) discloses the use of Tat peptides to transport covalently linked biologically active cargo molecules into the cytoplasm and nuclei of cells. Frankel only discloses covalently linked cargo moieties that are (therapeutic, diagnostic or prophylactic), and does not teach or suggest the attachment of molecules that facilitate diffusion, plasticity, neurite growth, and axon regeneration. These molecules can include but are not limited to molecules that overcome neurite out growth inhibition, or promote nerve growth such as soluble NOGO antagonists like NgR27-311, neural cell adhesion molecules like L1, neurotrophic factors, growth factors, phosphodiesterase inhibitors, and inhibitors of MAG or MOG. Additionally, deletion mutants may be combined with other compounds that promote remyelination such as neuregulins (GGF2) and antibodies that promote remyelination or proteoglycan degrading molecules to Tat peptides.
Regeneration following SCI is limited because of a variety of obstacles that include the deposition of CSPG at the glial scar, demyelination of axons, lack of trophic support and lack of axonal guidance. A single therapy directed against one aspect of SCI may not be as effective as a combinatorial approach. Fusion proteins with chondroitinase will allow combinatorial therapy with a single protein. Fusion partners for chondroitinase that will be constructed in this proposal were chosen from among proteins that have evidence for efficacy in SCI.
The use of a molecule that has the ability to both degrade extracellular matrix glycoproteins and to block or overcome the inhibitory nature of myelin components may be used to improve the ability of damaged neurons to grow or regenerate compared with either treatment alone. The proteoglycan degrading molecules may also be used advantageously to provide a method of facilitating access and diffusion of substances into cells or tissues through the use of at least one enzyme capable of cleaving proteoglycans and preferably degrading chondroitin sulfate proteoglycans (CSPG).
Embodiments of the present invention include compositions that comprise polypeptides which cleave proteoglycans, polypeptides that block and/or overcome the activity of neuronal growth inhibitory molecules, or a combination of these. The compositions containing the proteoglycan degrading molecule or neuronal growth inhibitory molecules may also include molecules for transduction of the polypeptides across cell membranes and the blood brain barrier. The compositions may be used in the treatment of spinal cord injuries and related disorders of the central nervous system (CNS). The compositions can be used in the regeneration of damaged neurological tissue and facilitate the diffusion and transport of therapeutic molecules capable of blocking and/or overcoming the activity of neuronal growth inhibitory molecules into damaged or diseased tissue.
Embodiments of the present invention include compositions and methods for their use to facilitate delivery and diffusion of therapeutics or diagnostic agents, and preferably agents that promote regeneration of nerves and axons, into cells or tissues. Preferably the composition includes the use of an enzyme capable of cleaving chondroitin sulfate proteoglycans (CSPG) to increase the diffusion of these agents into cells or tissues of the central nervous system.
Compositions of the present invention may include chimeric or fusion proteins capable of systemic use in the treatment of spinal cord injuries and related disorders of the central nervous system (CNS), and in particular, fusion proteins capable of crossing the blood brain barrier. The fusion protein may include a polypeptide transduction domain, a polypeptide domain capable of degrading a proteoglycan, preferably a domain cleaving chondroitin sulfate proteoglycan (CSPG), a polypeptide domain that blocks and or over comes the activity of neuronal growth inhibitory molecules, or any combination of these polypeptide domains that may be used in the treatment of spinal cord injuries and related disorders of the central nervous system (CNS). The various polypeptide domains may be linked or chemically bonded together by polypeptide linkers.
Compositions of the present invention include polynucleotides which encode for the chimeric or fusion proteins capable of systemic use in the treatment of spinal cord injuries and related disorders of the central nervous system (CNS), and in particular, they encode for fusion proteins capable of crossing the blood brain barrier. The polynucleotides which encode for these chimeric or fusion proteins may include a polynucleotide domain encoding for a polypeptide transduction domain, a polynucleotide domain encoding for a polypeptide domain capable of degrading a proteoglycan, preferably cleaving chondroitin sulfate proteoglycan (CSPG), a polynucleotide domain encoding for a polypeptide domain that blocks and or over comes the activity of neuronal growth inhibitory molecules, or any combination of these domains that may be used in the treatment of spinal cord injuries and related disorders of the central nervous system (CNS). The polynucleotide also includes one or more polynucleotide domains that encode for polypeptides that link the domains of the polypeptide together to form the fusion protein.
One embodiment of the present invention is a composition and a method for its use that facilitates the access and distribution of therapeutic and diagnostic agent in the composition into cells, through membranes or into tissues by the use of composition that includes at least one enzyme capable of cleaving proteoglycans, preferably the composition includes a fusion protein having an enzyme capable of cleaving CSPGs. The molecules or agents in the composition may include one or more of Growth factors including, Brain Derived Neurotrophic Factor, Insulin-like Growth Factor, Fibroblast Growth Factor, Ciliary Neurotrophic Factor, Glial Derived Neurotrophic Factor, Transforming Growth Factor, Glial Growth Factor 2, L1, GM1, Vascular Endothelial Growth Factor, Nerve Growth Factor, Immunophilins. Molecules in the composition can include fluorescent or contrast agents for imaging. The agents may include cells for transplant—stem cells, neurons, others, cells as delivery agents, chemotherapeutic agents, antibiotics, antibody therapies, Nogo receptor antagonists, other chondroitinase enzymes. The composition may include a transduction domain, an enzyme capable of cleaving proteoglycans, or both. Preferably the composition includes a fusion protein having a transduction domain, an enzyme domain capable of cleaving proteoglycans, or both. The fusion protein can facilitate the transport or modifies transport of such agents into cells, tissues, and/or otherwise inaccessible locations; and/or to enhance penetration rates, distance of penetration; or provide more even concentration distribution. Preferably the modified transport occurs through the use of at least one enzyme capable of cleaving CSPGs. The compositions can be used for treating a CNS injury, preferably the composition is used in the treatment of neuronal damage from a contusion injury.
Embodiments of the present invention include chimeric proteins of a proteoglycan degrading domain linked to a polypeptide that blocks the action of neuronal growth inhibitors such as but not limited to a Nogo-receptor antagonist (NgR27-311) domain or variant linked to a chondroitinase like chondroitinase ABC I or a variant of chondroitinase having one or more N terminal amino acids deleted. The compound may include chimeric proteins of a proteoglycan degrading domain linked to a polypeptide that is a neural cell adhesion promoter such as an L1 neural cell adhesion promoter domain or variant linked to chondroitinase ABC I or a variant of chondroitinase having one or more N terminal amino acids deleted. The chimeric proteins may include chimeric proteins of a proteoglycan degrading domain linked to a polypeptide that is a glial cell stimulator, such as but not limited to a GGF2 glial cell stimulator or variant linked to chondroitinase ABCI or a variant of chondroitinase having one or more N terminal amino acids deleted.
An E. Coli recombinant expression system and purification process can be used to produce essentially pure and catalytically active chondroitinase ABCI. These methods may be modified for producing chimeras of proteoglycan degrading molecules and other agents.
The chimera may be assayed for chondroitinase enzymatic activity and the specific biological activity of each fusion partner. Methods to measure the activities of the chimera may be modified for those used to measure chondroitinase activity including a spectrophotometric assay, zymography, an HPLC assay to detect CSPG disaccharide digestion products and an in vito neurite outgrowth assay. A neuron growth cone collapse assay can be used to evaluate NOGO receptor antagonists and a neurite outgrowth assay can be used measure L1 activity. GGF2 activity may be measured using a Schwann cell proliferation assay.
The compositions and method of the present invention can be used for the treatment of spinal cord injuries and in the promotion of regeneration of axons. The compositions of the present invention can also be used to promote plasticity, regrowth, repair, and/or regeneration of dysfunctional neurons in the CNS that have been damaged as a result of disease, such as degenerative diseases including Alzheimer's and Parkinson's disease. Advantageously, the use of proteoglycan degrading polypeptides or membrane transducing polypeptides in the compositions of the present invention also promote diffusion and access of damage or diseased tissue to other therapeutic agents promoting the regeneration of neurons.